The purpose of this grant is to analyze the function of the human TAN-1 gene in neoplasia and normal tissues. This gene was originally discovered spanning the site of a recurrent chromosomal breakpoint in human acute T lymphoblastic neoplasms. Analysis of normal, full length TAN-1 cDNA revealed strong sequence homology to the Drosophila gene Notch, which encodes a transmembrane protein that controls the choice of cell fates m numerous tissues of the developing fruit fly. Preliminary data indicate that insertion into bone marrow stem cells of TAN-1 cDNA fragments designed to mimic the gene broken by the translocation induces T lymphoblastic neoplasms in mice. Studies of the TAN-1 protein (tan-1) in normal cells show that it is proteolytically cleaved at a site just external to the transmembrane region and that the residual carboxy terminal polypeptide fragment closely resembles the truncated polypeptides produced from the translocated chromosome in neoplastic cells. Both the full length and processed tan-1 polypeptides are localized to the perinuclear cytoplasm, while the truncated product in neoplastic cells is predominantly intranuclear. The nuclear location of this polypeptide appears to be determined by a strong nuclear localization signal sequence within the intracellular domain of the protein. Additionally, the product of the BCL-3 proto-oncogene (bcl-3), which has recently been found to be involved in regulation of the transcription factor NFkappaB, binds to an intracellular region of tan-1 containing six so-called ankyrin repeats. Research proposed in this application will investigate four aspects of TAN-1 function, including the alternative possibilities of the developmental stage-specific transformation of T lymphocytes versus the autoregulation of Notch-like genes by truncated tan-1 polypeptides. The importance of nuclear localization for malignant transformation will be examined and an efficient in vitro assay system will be developed to study the role of different regions within tan-1 in the transforming ability of the protein. Ligands that bind to the extracellular domain of tan-1 will be sought and their genes molecularly cloned. The possible involvement of tan-1 in the NFkappaB pathway will be investigated using in vitro binding studies with tan-1, bcl-3, and NFkappaB and by a co-transfection of cells with TAN-1, BCL-3, and NFkappaB cDNAs along with a reporter gene behind a promoter normally regulated by NFkappaB.